Amino acid changes in Drosophila αPS2βPS integrins that affect ligand affinity

We developed a ligand-mimetic antibody Fab fragment specific for Drosophila αPS2βPS integrins to probe the ligand binding affinities of these invertebrate receptors. TWOW-1 was constructed by inserting a fragment of the extracellular matrix protein Tiggrin into the H-CDR3 of the αvβ3 ligand-mimetic antibody WOW-1. The specificity of αPS2βPS binding to TWOW-1 was demonstrated by numerous tests used for other integrin-ligand interactions. Binding was decreased in the presence of EDTA or RGD peptides and by mutation of the TWOW-1 RGD sequence or the βPS metal ion-dependent adhesion site (MIDAS) motif. TWOW-1 binding was increased by mutations in the αPS2 membrane-proximal cytoplasmic GFFNR sequence or by exposure to Mn²⁺. Although Mn²⁺ is sometimes assumed to promote maximal integrin activity, TWOW-1 binding in Mn²⁺ could be increased further by the αPS2 GFFNR→GFANA mutation. A mutation in the βPS I domain (βPS-b58; V409D) greatly increased ligand binding affinity, explaining the increased cell spreading mediated by αPS2βPS-b58. Further mutagenesis of this residue suggested that Val-409 normally stabilizes the closed head conformation. Mutations that potentially reduce interaction of the integrin β subunit plexin-semaphorin-integrin (PSI) and stalk domains have been shown to have activating properties. We found that complete deletion of the βPS PSI domain enhanced TWOW-1 binding. Moreover the PSI domain is dispensable for at least some other integrin functions because βPS-ΔPSI displayed an enhanced ability to mediate cell spreading. These studies establish a means to evaluate mechanisms and consequences of integrin affinity modulation in a tractable model genetic system.